The field of this invention is the area of molecular biology, and in particular the DNA sequence encoding Protein Phosphatase Methylesterase-1 (PME-1, formerly called p44A), recombinant vectors, and methods for recombinant production of PME-1 demethylase and its use in identifying compositions with inhibitory activity.
Protein phosphatase 2A (PP2A) is a highly conserved serine/threonine phosphatase involved in the regulation of a wide variety of enzymes, signal transduction pathways, and cellular events [Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508; Lee, T. H., et al. (1991) Cell 64:415-423; Mayer-Jaekel, R. E. et al. (1993) Cell 72:621-633; Sontag, E. S. et al. (1993) Cell 75:887-897; Uemura, T. et al. (1993) Genes Dev. 7:429-440]. The minimal structure thought to exist in vivo consists of a heterodimer between a catalytic 36 kDa subunit termed C and a constant regulatory 63 kDa subunit termed A [Kremmer, E. et al. (1997) Mol. Cell Biol. 17:1692-1701; Usui, H. et al. (1988) J. Biol. Chem. 263:3752-3761]. This heterodimer is often further complexed with one of several additional regulatory subunits termed B, Bxe2x80x2, and Bxe2x80x3 [Cohen, P. (1989) supra]. In PP2A heterotrimers, the A subunit binds to both the catalytic C and regulatory B-type subunits [Ruediger, R. et al. (1992) J. Virol. 68:123-129; Ruediger, R. et al. (1994) Mol. Cell Biol. 12:4872-4882]. In the case of the B subunit, it has been shown that one or more of the nine C subunit carboxy terminal amino acids are essential for heterotrimer formation [Ogris, E. et al. (1997) Oncogene 15:911-917]. In cells stably transformed by the middle tumor antigen (MT) of polyomavirus, MT is found in place of the B subunit in a small portion (xe2x88x9210%) [Ulug, et al. (1992) J. Virol. 66:1458-1467] of PP2A complexes [Pallas, D. C. et al. (1990) Cell 60:167-176]. MT/PP2A complex formation is important for MT-mediated transformation [Grussenmeyer, et al. (1987) J. Virol. 61:3902-3909; Pallas, et al. (1988) J. Virol. 62:3934-3940; Glenn, G. M. et al. (1995) J. Virol. 69:3729-3736; Campbell, K. S. et al. (1995) J. Virol. 69:3721-3728]. Unlike for B subunit, formation of PP2A heterotrimers containing MT does not require the last nine amino acid residues of the C subunit [Ogris, E. et al. (1997) supra]. The small tumor antigens (STs) of various papovaviruses also form complexes with the A and C subunits of PP2A [Pallas, D. C. et al. (1990) supra].
Consistent with the multiple important roles that PP2A plays in diverse pathways and cellular events, PP2A is highly regulated. The regulatory mechanisms include modulation by regulatory subunits or inhibitory proteins and modulation by post-translational modification of the C subunit. Subunit composition of the PP2A complex affects both catalytic activity and substrate specificity [Agostinis, P. et al. (1992) Eur. J. Biochem. 205:241-248; Favre, B. et al. (1994) J. Biol. Chem. 269:16311-16317; Scheidtmann, K. H. et al. (1991) Mol. Cell. Biol. 11:1996-2003; Sola, M. M. et al. (1991) Biochem. Biophys. Acta 1094:211-216]. In the case of B subunit, changes of up to 100 fold have been documented using cdc2 phosphorylated substrates [Agostinis, P. et al. (1992) Eur. J. Biochem. 205:241-248; Ferrigno, P. et al. (1993) Mol. Biol. Cell 4:669-677; Mayer-Jaekel, R. E. et al. (1994) Journal of Cell Science 107:2609-2618; Ogris, E. et al. (1997) supra; Sola, M. M. et al. (1991) Biochem. Biophys. Acta 1094:211-216]. Two PP2A inhibitor proteins have been reported: I1PP2A (also called PHAPI) and I2PP2A (also called PHAPII or SET) [Li, M. et al. (1996) Biochemistry 34:1988-1996; Li, M. et al. (1996) Biochemistry 35: 6998-7002; Li, M. et al. (1995) J. Biol. Chem. 271:11059-11062]. These also appear to be substrate-dependent in their effects. Perusal of the NCBI GenBank and EST databases via BLAST followed by sequence comparisons using DNASTAR MegAlign software indicates the existence of three different human PHAPI isoforms encoded by different genes and the presence of multiple alternatively spliced forms of PHAPII. A Xenopus homolog of PHAPII was recently shown to interact with B-type cyclins in vitro [Kellogg, D. R. et al. (1995) J. Cell Biol. 130:661-673], but the molecular consequences of this interaction in the regulation of PP2A are not known.
The post-translational modifications of the C subunit that have been reported to modulate PP2A activity include phosphorylation and methylation. Inhibition of PP2A activity in vitro was found upon C subunit phosphorylation at either tyrosine 307 or at one or more unidentified threonine residues [Chen, J. et al. (1992) Science 257:1261-1264; Guo, H. and Damuni, Z. (1993) Proc. Natl. Acad. Sci. USA 90:2500-2504]. A similar modification may occur in vivo in response to transformation or growth stimulation [Chen, J. et al. (1994) J. Biol. Chem. 269:7957-7962]. The first indication that PP2A C subunit was methylated involved two observations. A 36 kDa SV40 small tumor antigen (ST)-associated cellular protein is a major acceptor of the methyl group from radiolabeled S-adenosyl methionine added to cell extracts [Rundell, K (1987) J. Virol. 61:1240-1243]. This ST-associated cellular protein was reported to be the PP2A C subunit [Pallas, D. C. et al. (1990) supra]. The site of methylation of the PP2A C subunit has been identified as leucine 309 [Favre, B. et al. (1994) supra; Lee, J. and Stock, J. (1993) J. Biol. Chem. 268:19192-19195; Xie, H. and Clarke, S. (1994) J. Biol. Chem. 269:1981-1984]. One study reported an approximately two-fold increase in the activity of PP2A upon methylation, adjusting for the stoichiometry of methylation [Favre, B. et al. (1994) supra]. Only phosphorylase a and the peptide substrate, phosphorylated Kemptide, were used in that study. These substrates often give similar results. Thus, it remains to be determined whether greater effects might be observed with other substrates. Based on differential antibody recognition of methylated and non-methylated C subunit, PP2A has been reported to undergo cell cycle dependent changes in methylation [Turowski, P. et al. (1995) J. Cell Biol. 129:397-410]. It is not known whether methylation of PP2A affects the subunit composition of the enzyme. Partially purified fractions of PP2A containing A/C heterodimers or A/B/C heterotrimers have both been shown to be substrates for the PP2A methyltransferase [Xie, H. and Clarke, S. (1994) supra]. There are also data which indicate that methylated C subunit can associate with SV40 ST [Rundell, K. (1987) supra].
The B subunit functions in cell cycle progression through mitosis and in cytokinesis [Healy, A. M. et al. (1991) Mol. Cell Biol. 11:5767-5780; Mayer-Jaekel, R. E. et al. (1993) supra; Uemura, T. et al. (1993) Genes Dev. 7:429-440]. In cells stably transformed by the middle tumor antigen (MT) of polyomavirus, MT is found in place of the B subunit in a small portion (xcx9c10%) [Ulug, E. T. et al. supra] of PP2A complexes [Pallas, D. C. et al. (1990) supra]. MT/PP2A complex formation is known to be important for MT-mediated transformation [Campbell, K. S. et al. (1995) supra; Glenn, G. M. et al. (1995) supra; Grussenmeyer, T. et al. (1987) supra; Pallas, D. C. et al. (1988) supra], but the precise functional consequences of MT association with PP2A are still being elucidated. It was recently shown that there is a requirement for direct B/C subunit interaction to form stable heterotrimers [Ogris, E. et al. (1997) supra].
The nine carboxy-terminal amino acids of the PP2A C subunit, residues 301 to 309, include tyrosine 307, the site of phosphorylation in vitro by v-src, and two potential sites of threonine phosphorylation, residues 301 and 304. Seven of these nine residues, including threonine 304 and tyrosine 307, are found in every PP2A C subunit cloned to date. Threonine 301 is somewhat less conserved.
In order to study cellular proteins which interact with PP2A, two catalytically inactive C subunit mutants were generated and used to form stable complexes. The present invention describes the identification of one of these proteins, herein named Protein Phosphatase Methylesterase-1 (PME-1).
Due to the fact that PP2A is shown to regulate multiple cellular pathways by dephosphorylating several key proteins, there has been a long felt need in the art to understand the molecular mechanisms by which PP2A activity is modulated. The present invention describes cloning of one such modulating enzyme for human PP2A, named herein PME-1, and also shows how to produce recombinant PME-1 polypeptide, which is then used in in vitro assays to identify inhibitors for PME-1 activity.
It is an object of the present invention to provide nucleotide sequences encoding protein phosphatase methylesterase-1 (PME-1) and the deduced amino acid sequence therefor. Specifically exemplified coding sequences are given in Table 2, together with the deduced amino acid sequence for the human; Tables 6 and 3 for the yeast; Tables 7 and 4 for the nematode. All synonymous coding sequences for the exemplified amino acid sequences are within the scope of the present invention.
It is a further object of the present invention to provide functionally equivalent coding and protein sequences, including equivalent sequences from other mammals and other organisms, including but not limited to yeast and nematodes, and variant sequences from humans. Functionally equivalent PME-1 coding sequences are desirably from about 50% to about 80% nucleotide sequence homology (identity) to the specifically identified PME-1 coding sequence, from about 80% to about 95%, and desirably from about 95% to about 100% identical in coding sequence to the specifically exemplified coding sequence. Each integer and each subset of each specified range is intended within the context of the present invention.
Hybridization conditions of particular stringency provide for the identification of homologs of the human PME-1 coding sequence from other species and the identification of variant human sequences, where those homologs and/or variant sequences have at least (inclusively) 50 to 85%, 85 to 100% nucleotide sequence identity, 90 to 100%, or 95 to 100% nucleotide sequence identity.
The PME-1 coding sequence and methods of the present invention include the homologous coding sequences in organisms other than humans and mice. Methods can be employed to isolate the corresponding coding sequences (for example, from cDNA) from other organisms, including but not limited to other mammals, avian species, Saccharomyces and Caenorhabditis elegans useful in the methods of this invention using the sequences disclosed herein and experimental techniques well known to the art.
It will further be understood by those skilled in the art that other nucleic acid sequences besides those disclosed herein for the PME-1 coding sequence will function as coding sequences synonymous with the exemplified coding sequences. Nucleic acid sequences are synonymous if the amino acid sequences encoded by those nucleic acid sequences are the same. The degeneracy of the genetic code is well known to the art. For many amino acids, there is more than one nucleotide triplet which serves as the codon for a particular amino acid, and one of ordinary skill in the art understands nucleotide or codon substitutions which do not affect the amino acid(s) encoded.
Specifically included in this invention are PME-1 sequences from other organisms than those exemplified herein, which sequences hybridize to the PME-1 sequence disclosed under stringent conditions. Stringent conditions refer to conditions understood in the art for a given probe length and nucleotide composition and capable of hybridizing under stringent conditions means annealing to a subject nucleotide sequence, or its complementary strand, under standard conditions (i.e., high temperature and/or low salt content) which tend to disfavor annealing of unrelated sequences. As specifically exemplified, xe2x80x9cconditions of high stringencyxe2x80x9d means hybridization and wash conditions of 65xc2x0-68xc2x0 C., 0.1xc3x97SSC and 0.1% SDS (indicating about 95-100% nucleotide sequence identity/similarity). Hybridization assays and conditions are further described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.
As used herein, conditions of moderate (medium) stringency are those with hybridization and wash conditions if 50-65xc2x0 C., 1xc3x97SSC and 0.1% SDS (where a positive hybridization result reflects about 80-95% nucleotide sequence identity). Conditions of low stringency are typically those with hybridization and wash conditions of 40-50xc2x0 C., 6xc3x97SSC and 0.1% SDS (reflecting about 50-80% nucleotide sequence identity).
As used herein, all or part of a nucleotide sequence refers specifically to all continuous nucleotides of a nucleotide sequence, or e.g. 1000 continuous nucleotides, 500 continuous nucleotides, 100 continuous nucleotides, 25 continuous nucleotides, and 15 continuous nucleotides.
Where PME-1-homologous coding sequences are to be isolated from other organisms, one desirably uses nucleotide probes or primers from the most highly conserved regions of the PME-1 protein. For example, the skilled artisan desirably uses hybridization probes or PCR primers encoding the active site region (GHSMGGA, amino acids 154-160, SEQ ID NO:5, in the protein sequence) and a second highly conserved sequence within the protein [GQMQGK, amino acids 333-338, SEQ ID NO:5) to derive probe or primer sequences.
It is well known in the biological arts that certain amino acid substitutions may be made in protein sequences without affecting the function of the protein. Generally, conservative amino acid substitutions or substitutions of similar amino acids are tolerated without affecting protein function. Similar amino acids can be those that are similar in size and/or charge properties, for example, aspartate and glutamate, and isoleucine and valine, are both pairs of similar amino acids. Similarity between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22, pp. 345-352, which is incorporated by reference herein, provides frequency tables for amino acid substitutions which can be employed as a measure of amino acid similarity. Dayhoff et al.""s frequency tables are based on comparisons of amino acid sequences for proteins having the same function from a variety of evolutionarily different sources.
Also within the scope of the present invention are recombinant host cells and recombinant vectors carrying the PME-1 coding sequences of the present invention. Desirably, those coding sequences are operably linked to transcriptional and translational control sequences functional in the host cell into which the vectors are introduced and maintained.
Further provided by the present invention are methods for the recombinant production of a PME-1 protein. After a suitable vector in which a PME-1 coding sequence is operably linked to transcriptional and translational control sequences is introduced into a recombinant host cell of choice, the recombinant host cells are cultured under conditions where the PME-1 sequences are expressed. The PME-1 can then be recovered, if desired. It is understood that the vector and host cells are chosen for maintenance of the vector within the host cell. Similarly, the transcriptional and translational control sequences are chosen for function in the host cell of choice. The specifically exemplified human PME-1 sequence can be modified, for example, using polymerase chain reaction (PCR) technology by substituting synonymous codons according to the known codon usage of the chosen host cell so that expression of the coding sequence is maximized.